Directed editing of cellular RNA via nuclear delivery of CRISPR/CAS9

Abstract

Disclosed herein is a technology to perform programmable RNA editing at single-nucleotide resolution using RNA-targeting CRISPR/Cas9. This approach, which Applicants have termed “Cas9-directed RNA editing” or “CREDIT,” provides a means to reversibly alter genetic information in a temporal manner, unlike traditional CRISPR/Cas9 driven genomic engineering which relies on permanently altering DNA sequence.

What is claimed is: 
     
       1. A recombinant expression system for CRISPR/Cas-directed RNA editing of a target RNA comprising:
 (A) a nucleic acid sequence encoding a CRISPR/Cas RNA editing fusion protein comprising a nuclease-dead CRISPR associated endonuclease (dCas) fused to a catalytically active deaminase domain of Adenosine Deaminase acting on RNA 2 (ADAR2); and 
 (B) a nucleic acid sequence encoding an extended single guide RNA (esgRNA) comprising: (i) a short extension sequence of homology to the target RNA comprising a mismatch for a target adenosine, (ii) a dCas scaffold binding sequence, and (iii) a spacer sequence comprising a region of homology to the target RNA, wherein the sequences of the esgRNA (i), (ii), and (iii) are situated 3′ to 5′ in the esgRNA. 
 
     
     
       2. The recombinant expression system of  claim 1 , wherein (A) and (B) are comprised within the same vector or comprised within different vectors. 
     
     
       3. The recombinant expression system of  claim 1 , wherein the catalytically active deaminase domain of ADAR2 comprises an E488Q mutation. 
     
     
       4. The recombinant expression system of  claim 1 , wherein the dCas N-terminal domain is fused to the C-terminus of the catalytically active deaminase domain of ADAR2. 
     
     
       5. The recombinant expression system of  claim 1 , wherein the dCas is fused to the catalytically active deaminase domain of ADAR2 via a linker. 
     
     
       6. The recombinant expression system of  claim 5 , wherein the linker is a semi-flexible XTEN peptide linker. 
     
     
       7. The recombinant expression system of  claim 1 , wherein the short extension sequence of the esgRNA comprises a region of homology capable of near-perfect RNA-RNA base pairing with the target sequence. 
     
     
       8. The recombinant expression system of  claim 1 , wherein the esgRNA further comprises a marker sequence. 
     
     
       9. The recombinant expression system of  claim 1 , wherein the esgRNA further comprises a RNA polymerase III promoter sequence. 
     
     
       10. The recombinant expression system of  claim 9 , wherein the RNA polymerase III promoter sequence is a U6 promoter sequence. 
     
     
       11. The recombinant expression system of  claim 1 , wherein the esgRNA comprises a linker sequence between the spacer sequence and the scaffold sequence. 
     
     
       12. The recombinant expression system of  claim 2 , wherein at least one vector is a viral vector. 
     
     
       13. The recombinant expression system of  claim 12 , wherein the viral vector is an adeno-associated viral vector (AAV), lentiviral vector, or an adenoviral vector. 
     
     
       14. A vector comprising a nucleic acid encoding an extended single guide RNA (esgRNA) comprising (i) a short extension sequence of homology to a target RNA comprising a mismatch for a target adenosine, (ii) a dCas scaffold binding sequence, and (iii) a sequence complementary to the target sequence (spacer sequence), wherein (i), (ii) and (iii) are situated 3′ to 5′ in the esgRNA. 
     
     
       15. The vector of  claim 14 , wherein the vector is a viral vector. 
     
     
       16. The vector of  claim 15 , wherein the viral vector is an adeno-associated viral vector (AAV), lentiviral vector, or an adenoviral vector. 
     
     
       17. The vector of  claim 14 , further comprising an expression control element. 
     
     
       18. A viral particle comprising the vector of  claim 14 . 
     
     
       19. An isolated cell comprising the recombinant expression system of  claim 1 . 
     
     
       20. A kit comprising:
 (A) one or more selected from the group consisting of:
 (i) a recombinant expression system comprising:
 (a) a nucleic acid sequence encoding a CRISPR/Cas RNA editing fusion protein comprising a nuclease-dead CRISPR associated endonuclease (dCas) fused to a catalytically active deaminase domain of Adenosine Deaminase acting on RNA 2 (ADAR2); and 
 (b) a nucleic acid sequence encoding an extended single guide RNA (esgRNA) comprising:
 (i) a short extension sequence of homology to the target RNA comprising a mismatch for a target adenosine, 
 (ii) a dCas scaffold binding sequence, and 
 (iii) a spacer sequence comprising a region of homology to the target RNA, 
 
 wherein the sequences of the esgRNA (i), (ii), and (iii) are situated 3′ to 5′ in the esgRNA; 
 
 (ii) a vector comprising a nucleic acid encoding (A)(i)(a) and (b); 
 (iii) a vector comprising a nucleic acid encoding (A)(i)(b); 
 (iv) a viral particle comprising the recombinant expression system of (A)(i)(a) and (b); 
 (v) an isolated cell comprising the recombinant expression system of (A)(i)(a) and (b); and 
 (vi) an esgRNA comprising (i) a short extension sequence of homology to a target RNA comprising a mismatch for a target adenosine, and (ii) a dCas scaffold binding sequence, and (iii) a spacer sequence comprising a region of homology to the target RNA, wherein the sequences of the esgRNA (i), (ii), and (iii) are situated 3′ to 5′ in the esgRNA and 
 
 (B) instructions for use.